Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.
Molecular biology of the cell (Impact Factor: 4.47). 02/2011; 22(8):1191-206. DOI: 10.1091/mbc.E10-07-0599
Source: PubMed


Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase that directs mitotic progression by phosphorylation of a large number of substrate proteins. Cdk1 activation reaches the peak level due to positive feedback mechanisms. By inhibiting Cdk chemically, we showed that, in prometaphase, when Cdk1 substrates approach the peak of their phosphorylation, cells become capable of proper M-to-G1 transition. We interfered with the molecular components of the Cdk1-activating feedback system through use of chemical inhibitors of Wee1 and Myt1 kinases and Cdc25 phosphatases. Inhibition of Wee1 and Myt1 at the end of the S phase led to rapid Cdk1 activation and morphologically normal mitotic entry, even in the absence of G2. Dampening Cdc25 phosphatases simultaneously with Wee1 and Myt1 inhibition prevented Cdk1/cyclin B kinase activation and full substrate phosphorylation and induced a mitotic "collapse," a terminal state characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis. This was blocked by the PP1/PP2A phosphatase inhibitor, okadaic acid. These findings suggest that the positive feedback in Cdk activation serves to overcome the activity of Cdk-opposing phosphatases and thus sustains forward progression in mitosis.

Download full-text


Available from: Tamara A Potapova
  • Source
    • "Oil objective lens. Cells were then traced using Image J, and corrected total cell fluorescence (CTCF) values were calculated as described (Burgess et al., 2010; Gavet and Pines, 2010; Potapova et al., 2011; McCloy et al., 2014). At least 30 cells per coverslip were counted, and the experiment was repeated at least twice. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Glioblastoma's (GBM) extensive invasive capacity makes it resistant to surgery, radio- and chemotherapy, and thus makes it lethal. In vivo, GBM invasion is mediated by Rho GTPases through unidentified downstream effectors. mammalian Diaphanous (mDia) family formins are Rho-directed effectors that regulate the F-actin cytoskeleton to support tumor cell motility. Historically, anti-invasion strategies focused upon mDia inhibition, while activation remained unexplored. The recent development of small molecules directly inhibiting or activating mDia-driven F-actin assembly that supports motility allows for exploration of their role in GBM. We used the formin inhibitor SMIFH2 and mDia agonists IMM-01/-02 and mDia2-DAD peptides which disrupt autoinhibition to examine the roles of mDia inactivation verses activation in GBM cell migration and invasion in vitro, and in an ex vivo brain slice invasion model. Inhibiting mDia suppressed directional migration and spheroid invasion, while preserving intrinsic random migration. mDia agonism abrogated both random intrinsic and directional migration and halted U87 spheroid invasion in ex vivo brain slices. Thus, mDia agonism is a superior GBM anti-invasion strategy. We conclude that formin agonism impedes the most dangerous GBM component, tumor spread into surrounding healthy tissue. Formin activation impairs novel aspects of transformed cells and informs the development of anti-GBM invasion strategies.
    Full-text · Article · Sep 2015 · Molecular biology of the cell
  • Source
    • "To determine the total fluorescence detected in the z-stack, a region was drawn around each cell to be measured, and three regions next to the selected cell that had no fluorescence were used for background subtraction. The corrected total cell fluorescence for each cell was calculated using the following formula (Gavet and Pines, 2010; Potapova et al., 2011): "
    [Show abstract] [Hide abstract]
    ABSTRACT: Algal growth is strongly affected by nitrogen (N) availability. Diatoms, an ecologically important group of unicellular algae, have evolved several acclimation mechanisms to cope with N deprivation. In this study, we integrated physiological data with transcriptional and metabolite data to reveal molecular and metabolic modifications in N-deprived conditions in the marine diatom Phaeodactylum tricornutum. Physiological and metabolite measurements indicated that the photosynthetic capacity and chlorophyll content of the cells decreased, while neutral lipids increased in N-deprived cultures. Global gene expression analysis showed that P. tricornutum responded to N deprivation through an increase in N transport, assimilation, and utilization of organic N resources. Following N deprivation, reduced biosynthesis and increased recycling of N compounds like amino acids, proteins, and nucleic acids was observed at the transcript level. The majority of the genes associated with photosynthesis and chlorophyll biosynthesis were also repressed. Carbon metabolism was restructured through downregulation of the Calvin cycle and chrysolaminarin biosynthesis, and co-ordinated upregulation of glycolysis, the tricarboxylic acid cycle, and pyruvate metabolism, leading to funnelling of carbon sources to lipid metabolism. Finally, reallocation of membrane lipids and induction of de novo triacylglycerol biosynthesis directed cells to accumulation of neutral lipids. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.
    Full-text · Article · Sep 2015 · Journal of Experimental Botany
  • Source
    • "The number of Mk was quantitated by counting Mk in 10 low power fields (16X objective).10 The corrected total cell fluorescence for cMpl expression per MK (6 MK/Bm sample were considered) was quantitated by ImageJ software.11,12 With the exception of samples from MDS patients, cMpl quantitation was performed on mature Mk as conventionally defined.13 "
    [Show abstract] [Hide abstract]
    ABSTRACT: The relationship between thrombopoietin (TPO) and its receptor cMpl in thrombocytopenic conditions has not been entirely clarified. To elucidate this interplay may expand the spectrum of indications of TPO mimetics. In this study we have explored the relationship between TPO and cMpl in platelets and megakaryocytes of 43 patients with thrombocytopenia due to idiopathic thrombocytopenic purpura (ITP), bone marrow hypoplasia, myelodysplastic syndromes (MDS), and familial thrombocytopenia. Data were compared to cMpl and TPO in patients with a normal platelet count and in patients with thrombocytosis due to essential thrombocythemia (ET). All but familial patients showed higher TPO compared to controls. All thrombocytopenic states were invariably associated with increased expression of platelet cMPL compared to healthy controls. ET patients showed normal TPO and a trend toward a reduced cMpl expression. Immunofluorescence of bone marrow sections from patients with ITP and MDS failed to show a peculiar pattern compared to controls. Multiple mechanisms regulate TPO and cMpl in thrombocytopenic conditions.
    Full-text · Article · Jan 2014 · Hematology Reports
Show more